1Gladstone Institute of Neurological Disease, University of California, San Francisco, California 94158, USA.

Abstract

The accumulation of amyloid-beta (Abeta) peptides in the brain of patients with Alzheimer's disease (AD) may arise from an imbalance between Abeta production and clearance. Overexpression of the Abeta-degrading enzyme neprilysin in brains of human amyloid precursor protein (hAPP) transgenic mice decreases overall Abeta levels and amyloid plaque burdens. Because AD-related synaptic and cognitive deficits appear to be more closely related to Abeta oligomers than to plaques, it is important to determine whether increased neprilysin activity also diminishes the levels of pathogenic Abeta oligomers and related neuronal deficits in vivo. To address this question, we crossed hAPP transgenic mice with neprilysin transgenic mice and analyzed their offspring. Neprilysin overexpression reduced soluble Abeta levels by 50% and effectively prevented early Abeta deposition in the neocortex and hippocampus. However, it did not reduce levels of Abeta trimers and Abeta*56 or improve deficits in spatial learning and memory. The differential effect of neprilysin on plaques and oligomers suggests that neprilysin-dependent degradation of Abeta affects plaques more than oligomers and that these structures may form through distinct assembly mechanisms. Neprilysin's inability to prevent learning and memory deficits in hAPP mice may be related to its inability to reduce pathogenic Abeta oligomers. Reduction of Abeta oligomers will likely be required for anti-Abeta treatments to improve cognitive functions.

NEP overexpression reduces Aβ levels and prevents early plaque deposition without altering levels of C-terminal APP fragments. A,B Neprilysin overexpression in NEP and hAPP-J20/NEP mice was confirmed by western blot analysis of membrane fractions isolated from cortex-plus tissues (see Methods) of 2–4-month-old mice. Tubulin was used as a loading control. A, Representative western blot. B, Densitometric quantitation of western blot signals revealed that NEP levels were 11-fold higher in NEP and hAPP-J20/NEP mice than in NTG controls and that endogenous NEP levels in hAPP-J20 mice and NTG controls were similar. Numbers in bars represent numbers of mice analyzed per group. C, Particulate membrane fractions were isolated from cortex-plus tissues of 5–6-month-old hAPP-J20 and hAPP-J20/NEP mice and analyzed by western blotting with an antibody (CT-15) against hAPP C-terminal fragments (CTFs). Tubulin was used as a loading control. Labels on right identify bands putatively representing phosphorylated or unphosphorylated C83, C89, or C99. The rightmost lane contains a sample from a TG mouse of line I5, which expresses wildtype hAPP (Mucke et al., 2000); it illustrates that this assay was sensitive enough to detect the effect of the Swedish mutation in hAPP-J20 on CTF production. D, Aβ levels in hippocampal (Hip) and cortex-plus (Ctx) tissues of 2–4-month-old hAPP-J20 and hAPP-J20/NEP mice without plaques were determined by ELISA. E,F Levels of Aβ monomers in 6–7-month-old hAPP-J20 and hAPP-J20/NEP mice were compared by western blot analysis of TBST fractions isolated from whole hemibrains. E, Representative western blot. F, Densitometric quantitation of western blot signals. G,H Aβ deposits in hippocampal sections of 4–6-month-old hAPP-J20 and hAPP-J20/NEP mice were detected by immunostaining with the 3D6 antibody. I,J, Quantitation of percent area occupied by Aβ-immunoreactive deposits (I) and number of deposits (J). *p < 0.05, **p < 0.01, ***p < 0.001 vs hAPP mice (Student’s t test). ###p < 0.001 vs hAPP and NTG mice (Tukey’s test). Values are mean ± SEM from mice on the C57BL/6J background.

Overexpression of NEP does not reduce levels of Aβ*56 or Aβ trimers. Different Aβ assemblies were measured in 5–6-month-old hAPPFAD TG C57BL/6J mice from line J20 and from the lower expresser line J9. A, Representative western blot of membrane-enriched fractions from combined hippocampal and cortex-plus lysates probed with the anti-Aβ antibody 6E10, which recognizes Aβ*56 and hAPP, as indicated on right. B, Aβ trimers in soluble fractions of combined hippocampal and cortex-plus lysates were immunoprecipitated with the anti-Aβ antibody 4G8 and detected by western blotting with the anti-Aβ antibody 3D6. C, D, Relative levels of Aβ*56 (C) and Aβ trimers (D) were determined by densitometric analysis of western blot signals. E, Representative western blot of TBS and guanidine fractions isolated from hemibrains of 3–17-month-old hAPP-J20 mice. Aβ monomers and dimers were immunoprecipitated with the 6E10 antibody and detected by western blotting with the 3D6 antibody. Note that dimers were detected only in 17-month-old mice. Similar results were obtained with TBST fractions (data not shown). **p < 0.01, ***p < 0.001 vs hAPP-J20 and hAPP-J20/NEP mice (Tukey’s test). Values are means ± SEM.